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Identification of a Domain of Axin That Binds to the Serine/Threonine Protein Phosphatase 2A and a Self-binding Domain

1999; Elsevier BV; Volume: 274; Issue: 6 Linguagem: Inglês

10.1074/jbc.274.6.3439

1083-351X

Wei Hsu, Li Zeng, Frank Costantini,

Skin and Cellular Biology Research

Axin is a negative regulator of embryonic axis formation in vertebrates, which acts through a Wnt signal transduction pathway involving the serine/threonine kinase GSK-3 and β-catenin. Axin has been shown to have distinct binding sites for GSK-3 and β-catenin and to promote the phosphorylation of β-catenin and its consequent degradation. This provides an explanation for the ability of Axin to inhibit signaling through β-catenin. In addition, a more N-terminal region of Axin binds to adenomatous polyposis coli (APC), a tumor suppressor protein that also regulates levels of β-catenin. Here, we report the results of a yeast two-hybrid screen for proteins that interact with the C-terminal third of Axin, a region in which no binding sites for other proteins have previously been identified. We found that Axin can bind to the catalytic subunit of the serine/threonine protein phosphatase 2A through a domain between amino acids 632 and 836. This interaction was confirmed by in vitro binding studies as well as by co-immunoprecipitation of epitope-tagged proteins expressed in cultured cells. Our results suggest that protein phosphatase 2A might interact with the Axin·APC·GSK-3·β-catenin complex, where it could modulate the effect of GSK-3 on β-catenin or other proteins in the complex. We also identified a region of Axin that may allow it to form dimers or multimers. Through two-hybrid and co-immunoprecipitation studies, we demonstrated that the C-terminal 100 amino acids of Axin could bind to the same region as other Axin molecules. Axin is a negative regulator of embryonic axis formation in vertebrates, which acts through a Wnt signal transduction pathway involving the serine/threonine kinase GSK-3 and β-catenin. Axin has been shown to have distinct binding sites for GSK-3 and β-catenin and to promote the phosphorylation of β-catenin and its consequent degradation. This provides an explanation for the ability of Axin to inhibit signaling through β-catenin. In addition, a more N-terminal region of Axin binds to adenomatous polyposis coli (APC), a tumor suppressor protein that also regulates levels of β-catenin. Here, we report the results of a yeast two-hybrid screen for proteins that interact with the C-terminal third of Axin, a region in which no binding sites for other proteins have previously been identified. We found that Axin can bind to the catalytic subunit of the serine/threonine protein phosphatase 2A through a domain between amino acids 632 and 836. This interaction was confirmed by in vitro binding studies as well as by co-immunoprecipitation of epitope-tagged proteins expressed in cultured cells. Our results suggest that protein phosphatase 2A might interact with the Axin·APC·GSK-3·β-catenin complex, where it could modulate the effect of GSK-3 on β-catenin or other proteins in the complex. We also identified a region of Axin that may allow it to form dimers or multimers. Through two-hybrid and co-immunoprecipitation studies, we demonstrated that the C-terminal 100 amino acids of Axin could bind to the same region as other Axin molecules. Axin, the product of the mouse gene originally calledFused (1Reed S.C. Genetics. 1937; 22: 1-13Crossref PubMed Google Scholar), has been shown to negatively regulate an early step in vertebrate embryonic axis formation, through its ability to modulate a Wnt signal transduction pathway (2Zeng L. Fagotto F. Zhang T. Hsu W. Vasicek T.J. Perry III, W.L. Lee J.J. Tilghman S.M. Gumbiner B.M. Costantini F. Cell. 1997; 90: 181-192Abstract Full Text Full Text PDF PubMed Scopus (779) Google Scholar). The Fusedallele (Axin Fu), as well as two other spontaneous alleles, Kinky (Axin Ki) andKnobbly (Axin Kb), caused similar dominant phenotypes characterized mainly by kinking and shortening of the tail (1Reed S.C. Genetics. 1937; 22: 1-13Crossref PubMed Google Scholar, 3Gluecksohn-Schoenheimer S. J. Exp. Zool. 1949; 110: 47-76Crossref PubMed Scopus (73) Google Scholar, 4Jacobs-Cohen R.J. Spiegelman M. Cookingham J.C. Bennett D. Genet. Res. 1984; 43: 43-50Crossref PubMed Scopus (33) Google Scholar). Axin Ki and Axin Kb also caused recessive embryonic lethality at E8–E10. A fourth allele,Axin Tgl, induced by a random transgene insertion, had no dominant effects but caused recessive lethal embryonic defects similar to those observed in Axin Ki/Ki orAxin Kb/Kb embryos (5Perry W.L.I. Vasicek T.J. Lee J.J. Rossi J.M. Zeng L. Zhang T. Tilghman S.M. Costantini F. Genetics. 1995; 141: 321-332Crossref PubMed Google Scholar). Embryos homozygous for any of the recessive lethal alleles showed frequent neuroectodermal defects, including truncation or incomplete closure of the anterior neural folds, as well as cardiac defects. An intriguing feature of many homozygous embryos was a duplication of the embryonic axis, suggesting a role for Axin in embryonic axial development (3Gluecksohn-Schoenheimer S. J. Exp. Zool. 1949; 110: 47-76Crossref PubMed Scopus (73) Google Scholar, 5Perry W.L.I. Vasicek T.J. Lee J.J. Rossi J.M. Zeng L. Zhang T. Tilghman S.M. Costantini F. Genetics. 1995; 141: 321-332Crossref PubMed Google Scholar,6Tilghman S.M. Genome Res. 1996; 6: 773-780Crossref PubMed Scopus (15) Google Scholar). With the aid of the Axin Tgl insertional allele, the gene was cloned, and the wild type and mutant Axin alleles were characterized (2Zeng L. Fagotto F. Zhang T. Hsu W. Vasicek T.J. Perry III, W.L. Lee J.J. Tilghman S.M. Gumbiner B.M. Costantini F. Cell. 1997; 90: 181-192Abstract Full Text Full Text PDF PubMed Scopus (779) Google Scholar, 5Perry W.L.I. Vasicek T.J. Lee J.J. Rossi J.M. Zeng L. Zhang T. Tilghman S.M. Costantini F. Genetics. 1995; 141: 321-332Crossref PubMed Google Scholar, 7Vasicek T.J. Zeng L. Guan X.J. Zhang T. Costantini F. Tilghman S.M. Genetics. 1997; 147: 777-786Crossref PubMed Google Scholar). The murine Axin gene is ubiquitously expressed in wild type embryos and adult tissues, encoding a major mRNA of ∼4 kb 1The abbreviations used are: kb, kilobase pair(s); APC, adenomatous polyposis coli; PP2A, protein phosphatase 2A; GST, glutathione S-transferase; RGS, regulation of G-protein signaling; CMV, cytomegalovirus; PAGE, polyacrylamide gel electrophoresis; HA, hemagglutinin.1The abbreviations used are: kb, kilobase pair(s); APC, adenomatous polyposis coli; PP2A, protein phosphatase 2A; GST, glutathione S-transferase; RGS, regulation of G-protein signaling; CMV, cytomegalovirus; PAGE, polyacrylamide gel electrophoresis; HA, hemagglutinin.and a minor 3-kb mRNA. The ∼4-kb mRNA is found in two isoforms that encode proteins of 956 (form 1) and 992 amino acids (form 2). Form 2 is identical to form 1 except for an insertion of 36 amino acids at position 856, due to alternative splicing. The Axin sequence revealed two regions of homology to other protein families as follows: an RGS domain (8De Vries L. Elenko E. Hubler L. Jones T.L. Farquhar M.G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 15203-15208Crossref PubMed Scopus (155) Google Scholar, 9Koelle M.R. Horvitz H.R. Cell. 1996; 84: 115-125Abstract Full Text Full Text PDF PubMed Scopus (472) Google Scholar) at amino acids 213–338 and a "DIX domain" (10Cadigan K.M. Nusse R. Genes Dev. 1997; 11: 3286-3305Crossref PubMed Scopus (2206) Google Scholar) at the extreme C terminus. The RGS domains of bona fide RGS (Regulation of G-protein Signaling) proteins bind to GαaI proteins and serve as a GTPase-activating proteins (reviewed in Ref. 11Dohlman H.G. Thorners J. J. Biol. Chem. 1997; 272: 3871-3874Abstract Full Text Full Text PDF PubMed Scopus (445) Google Scholar). However, the Axin RGS domain differed from the consensus at most of the residues that make important contacts with the Giαa switch regions (12Tesmer J.J. Berman D.M. Gilman A.G. Sprang S.R. Cell. 1997; 89: 251-261Abstract Full Text Full Text PDF PubMed Scopus (677) Google Scholar), suggesting that it probably has a different function. The DIX domain is a region of similarity between the N terminus of Disheveled proteins (Drosophila Dsh and its vertebrate homologs) and the C terminus of Axin (10Cadigan K.M. Nusse R. Genes Dev. 1997; 11: 3286-3305Crossref PubMed Scopus (2206) Google Scholar). Whereas truncation of a 165-amino acid N-terminal segment of Dsh, including this domain, abolished its activity in a Drosophila cell culture assay for Wingless signaling (13Yanagawa S. van Leeuwen F. Wodarz A. Klingensmith J. Nusse R. Genes Dev. 1995; 9: 1087-1097Crossref PubMed Scopus (339) Google Scholar), the specific role of this domain remains obscure. Thus, the sequence of Axin provided few clues as to its function. In both the Axin Tgl and Axin Kballeles, synthesis of the full-length mRNAs is precluded by a transgene insertion in the former (2Zeng L. Fagotto F. Zhang T. Hsu W. Vasicek T.J. Perry III, W.L. Lee J.J. Tilghman S.M. Gumbiner B.M. Costantini F. Cell. 1997; 90: 181-192Abstract Full Text Full Text PDF PubMed Scopus (779) Google Scholar, 5Perry W.L.I. Vasicek T.J. Lee J.J. Rossi J.M. Zeng L. Zhang T. Tilghman S.M. Costantini F. Genetics. 1995; 141: 321-332Crossref PubMed Google Scholar) and a retroviral insertion in the latter (7Vasicek T.J. Zeng L. Guan X.J. Zhang T. Costantini F. Tilghman S.M. Genetics. 1997; 147: 777-786Crossref PubMed Google Scholar). As both of these alleles caused axial duplication, it was suggested that Axin normally plays a negative regulatory role in the response to an axis-inducing signal in early mouse embryogenesis. This hypothesis was supported by the ability of Axin mRNA to block dorsal axis formation, i.e. to "ventralize," when injected into early Xenopus embryos. Further analyses revealed that this ability is due to the inhibitory effect of Axin on a Wnt signaling pathway required for dorsal axis formation (2Zeng L. Fagotto F. Zhang T. Hsu W. Vasicek T.J. Perry III, W.L. Lee J.J. Tilghman S.M. Gumbiner B.M. Costantini F. Cell. 1997; 90: 181-192Abstract Full Text Full Text PDF PubMed Scopus (779) Google Scholar). This signaling pathway, which is closely related to the wingless signaling pathway of Drosophila, includes GSK-3, a serine/threonine kinase also involved in glycogen metabolism, and β-catenin, a protein also involved in cell adhesion (reviewed in Ref.14Miller J.R. Moon R.T. Genes Dev. 1996; 10: 2527-2539Crossref PubMed Scopus (606) Google Scholar). When active, GSK-3 can phosphorylate β-catenin (15Yost C. Torres M. Miller J.R. Huang E. Kimelman D. Moon R.T. Genes Dev. 1996; 10: 1443-1454Crossref PubMed Scopus (1009) Google Scholar), leading to its degradation through the ubiquitin-dependent proteolysis system (16Aberle H. Bauer A. Stappert J. Kispert A. Kemler R. EMBO J. 1997; 16: 3797-3804Crossref PubMed Scopus (2122) Google Scholar). In the presence of certain Wnts, GSK-3 is inhibited (through an unknown mechanism involving the cytoplasmic protein Dsh), allowing β-catenin to accumulate in the cytosol and to interact with transcription factors of the LEF/Tcf family (17Molenaar M. van de Wetering M. Oosterwegel M. Peterson-Maduro J. Godsave S. Korinek V. Roose J. Destree O. Clevers H. Cell. 1996; 86: 391-399Abstract Full Text Full Text PDF PubMed Scopus (1592) Google Scholar, 18Riese J. Yu X. Munnerlyn A. Eresh S. Hsu S.C. Grosschedl R. Bienz M. Cell. 1997; 88: 777-787Abstract Full Text Full Text PDF PubMed Scopus (391) Google Scholar). β-Catenin and LEF/Tcf then translocate to the nucleus, where they bind to DNA and activate target genes, which, in the early Xenopus embryo, include the homeobox gene Siamois (19Fagotto F. Guger K. Gumbiner B.M. Development. 1997; 124: 453-460PubMed Google Scholar, 20Carnac G. Kodjabachian L. Gurdon J.B. Lemaire P. Development. 1996; 122: 3055-3065PubMed Google Scholar, 21Brannon M. Kimelman D. Dev. Biol. 1996; 180: 344-347Crossref PubMed Scopus (122) Google Scholar). Through co-injection experiments, Axin was found to inhibit this signaling pathway at a level downstream of Wnt, Dsh, and GSK-3 but upstream of β-catenin and Siamois. Thus, it was proposed that Axin, directly or indirectly, stimulated the phosphorylation of β-catenin by GSK-3 (2Zeng L. Fagotto F. Zhang T. Hsu W. Vasicek T.J. Perry III, W.L. Lee J.J. Tilghman S.M. Gumbiner B.M. Costantini F. Cell. 1997; 90: 181-192Abstract Full Text Full Text PDF PubMed Scopus (779) Google Scholar). This prediction has been supported by several recent studies, which showed that Axin binds directly to GSK-3 and β-catenin, through distinct domains at amino acids 477–561 and 561–630, respectively (22Hart M.J. de los Santos R. Albert I.N. Rubinfeld B. Polakis P. Curr. Biol. 1998; 8: 573-581Abstract Full Text Full Text PDF PubMed Google Scholar, 23Itoh K. Krupnik V.E. Sokol S.Y. Curr. Biol. 1998; 8: 591-594Abstract Full Text Full Text PDF PubMed Google Scholar, 24Ikeda S. Kishida S. Yamamoto H. Murai H. Koyama S. Kikuchi A. EMBO J. 1998; 17: 1371-1384Crossref PubMed Scopus (1087) Google Scholar, 25Sakanaka C. Weiss J.B. Williams L.T. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3020-3023Crossref PubMed Scopus (282) Google Scholar). By simultaneously binding GSK-3β and β-catenin, Axin appears to promote the phosphorylation of β-catenin on serine/threonine residues. Axin also binds to APC, another protein implicated in the regulation of β-catenin (26Polakis P. Biochim. Biophys. Acta. 1997; 1332: F127-F147PubMed Google Scholar, 27Munemitsu S. Albert I. Souza B. Rubinfeld B. Polakis P. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 3046-3050Crossref PubMed Scopus (947) Google Scholar, 28Rubinfeld B. Albert I. Porfiri E. Fiol C. Munemitsu S. Polakis P. Science. 1996; 272: 1023-1026Crossref PubMed Scopus (1275) Google Scholar), through its RGS domain (22Hart M.J. de los Santos R. Albert I.N. Rubinfeld B. Polakis P. Curr. Biol. 1998; 8: 573-581Abstract Full Text Full Text PDF PubMed Google Scholar). The role of APC binding in the function of Axin remains unclear; a truncated Axin lacking the entire N terminus, including the RGS domain, still promoted the turnover of β-catenin (22Hart M.J. de los Santos R. Albert I.N. Rubinfeld B. Polakis P. Curr. Biol. 1998; 8: 573-581Abstract Full Text Full Text PDF PubMed Google Scholar) in mammalian cells, whereas an internal deletion of only the RGS domain abolished the ventralizing ability of Axin in Xenopusembryos (2Zeng L. Fagotto F. Zhang T. Hsu W. Vasicek T.J. Perry III, W.L. Lee J.J. Tilghman S.M. Gumbiner B.M. Costantini F. Cell. 1997; 90: 181-192Abstract Full Text Full Text PDF PubMed Scopus (779) Google Scholar). So far, the role of the C-terminal third of Axin (beyond the β-catenin binding region) is unclear. To identify proteins that interact with this region of Axin, we performed a yeast two-hybrid screen using the C-terminal 324 amino acids. We have thus identified a region of Axin that binds to the serine-threonine phosphatase PP2A. Our results suggest that PP2A may interact with the complex containing Axin, APC, GSK-3 and β-catenin, where it could serve to antagonize the effects of the kinase GSK-3. We also identified a C-terminal region of Axin that can bind to itself, suggesting that the protein may exist as a dimer or multimer. The bait plasmid pGBT9-Axin-(632–956) was constructed by inserting the PstI fragment of Axin cDNA (form 1) into the PstI site of pGBT9 vector (CLONTECH) to generate a Gal4DB-Axin fusion protein in yeast. This plasmid was sequenced to confirm that the coding sequences for Gal4DB and Axin were in frame. A series of pGBT9-Axin plasmids containing different regions of the Axin cDNA (Fig. 5) were created using convenient restriction enzyme sites. A method to create unidirectional nested deletions of double-stranded DNA clones using exonuclease III and mung bean nuclease was also performed to generate pGBT9-Axin-(632–910) and pGBT9-Axin-(632–836) (Exo-Size Deletion Kit, New England Biolabs). To express N-terminal FLAG-tagged Axin proteins in 293T cell, DNA fragments containing different regions of FLAG-Axin cDNA were cloned into a mammalian expression vector containing a CMV promoter (pcDNA3, Invitrogen). The pCMVT7-p36 plasmid contains a full-length PP2Ac cDNA that is tagged at the N terminus with T7 and inserted into the pCDNA3 vector. The pCMVHA-PR65 plasmid contains a full-length regulatory A subunit of PP2A tagged with HA at N terminus (29Hemmings B.A. Adams-Pearson C. Maurer F. Muller P. Goris J. Merlevede W. Hofsteenge J. Stone S.R. Biochemistry. 1990; 29: 3166-3173Crossref PubMed Scopus (188) Google Scholar). Three fragments of Axin cDNA were cloned downstream of the glutathioneS-transferase (GST) gene to generate pGST-Axin-(421–810), pGST-Axin-(632–810), and pGST-Axin-(632–956) plasmids and to produce recombinant proteins. The PP2Ac cDNA was inserted into a vector containing a translation initiation sequence and a FLAG-tag sequence to create the pBFT4-PP2Ac plasmid for in vitro transcription and translation. pGADNOT-PP2Accontains a full-length mouse PP2Ac cDNA cloned into a pGADNOT prey vector (30Dunaief J.L. Strober B.E. Guha S. Khavari P.A. Alin K. Luban J. Begemann M. Crabtree G.R. Goff S.P. Cell. 1994; 79: 119-130Abstract Full Text PDF PubMed Scopus (550) Google Scholar). The pGAD424-Axin-(194–956) plasmid contains an Axin cDNA fragment encoding amino acids 194–956 inserted into the pGAD424 prey vector (CLONTECH). The pGAD-PR65 plasmid contains a cDNA fragment encoding the full-length regulatory A subunit of PP2A cloned into the pGAD prey vector (31James P. Halladay J. Craig E.A. Genetics. 1996; 144: 1425-1436Crossref PubMed Google Scholar). The pGBT9-Axin-(632–956) plasmid was co-transformed with an expression library consisting of cDNAs from a murine macrophage cell line WEHI-3, which were cloned into the pGADNOT prey vector (30Dunaief J.L. Strober B.E. Guha S. Khavari P.A. Alin K. Luban J. Begemann M. Crabtree G.R. Goff S.P. Cell. 1994; 79: 119-130Abstract Full Text PDF PubMed Scopus (550) Google Scholar), into the yeast Y190 strain. Yeast transformants were grown on synthetic medium lacking leucine, tryptophan, and histidine. The expression of his andlacZ reporter genes were used to assay for clones encoding proteins that associate with Axin. The positive colonies were then grown on synthetic medium lacking only leucine to lose the pGBT9-Axin-(632–956) plasmid and to test for absence of β-galactosidase activity in the absence of the bait vector. To recover the pGADNOT plasmids, extracts of plasmid DNA from yeast were introduced into Escherichia coli JM83 by electroporation. Each of the pGADNOT plasmids was then co-transformed with pGBT-Axin-(632–956) plasmid into yeast Y190. Transformants were grown on synthetic medium lacking leucine and tryptophan and tested the ability to bind Axin in the β-galactosidase filter assay. Inserts of cDNA clones showing interaction with pGBT9-Axin-(632–956) were sequenced. pGST-Axin-(421–810), pGST-Axin-(632–810), and pGST-Axin-(632–956) plasmids were used to express and purify recombinant GST fusion proteins as described previously (32Hsu W. Kerppola T.K. Chen P.L. Curran T. Chen-Kiang S. Mol. Cell. Biol. 1994; 14: 268-276Crossref PubMed Scopus (181) Google Scholar). PP2Ac RNA was transcribedin vitro from pBFT4-PP2Ac using T7 RNA polymerase. PP2Ac protein was translated with reticulocyte lysate and labeled with [35S]methionine in vitro (Promega). In vitro binding of GST-Axin and PP2Ac was essentially as described (32Hsu W. Kerppola T.K. Chen P.L. Curran T. Chen-Kiang S. Mol. Cell. Biol. 1994; 14: 268-276Crossref PubMed Scopus (181) Google Scholar). The labeled PP2Ac proteins were incubated with GST or GST-Axin proteins in the association buffer, and protein complexes were precipitated with glutathione-Sepharose and analyzed by SDS-PAGE and autoradiography. 293T cells were transfected by a calcium phosphate-mediated transfection method (33Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning, A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989: 16.32-16.36Google Scholar) at 24 h after plating (2.0 × 106 cells per 100-mm dish). Ten mg of each plasmid DNA was used in each reaction, and sonicated salmon sperm DNA was used as a supplement to maintain the same DNA concentration in each transfection. Approximately 48 h after transfection, cells were lysed for protein expression and binding analyses. Protein extracts or immunoprecipitated complexes were subject to immunoblotting as described (34Harlow E. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1988: 471-510Google Scholar). Anti-FLAG (Kodak), anti-T7 conjugated with alkaline phosphatase (Novagen), or anti-Myc (Calbiochem) monoclonal antibodies were used to analyze the presence of tagged proteins in transfected cells. Except for anti-T7, bound antibodies were then detected with a goat anti-mouse IgG alkaline phosphatase-conjugated secondary antibody (American Qualex) and visualized by the chromogenic substrate reaction. 293T cells were lysed in a buffer containing 50 mm Tris-HCl (pH 8.0), 200 mm NaCl, 20 mm NaF, 20 mmβ-glycerophosphate, 100 mm sodium vanadate, 0.1 mm phenylmethylsulfonyl fluoride, 1 mmdithiothreitol, 2 mg/ml leupeptin, 2 mg/ml antipain, 0.1% Triton X-100, and 0.5% Nonidet P-40. Protein complexes were immunoprecipitated with monoclonal anti-FLAG or polyclonal anti-PP2Ac antibody (Promega), followed by immunoblotting analysis. A cDNA sequence encoding a C-terminal segment of Axin (amino acids 632–956, form 1) was cloned into the pGBT9 bait vector to produce a Gal4-DNA-binding domain-Axin fusion protein for yeast two-hybrid screening (35Fields S. Song O. Nature. 1989; 340: 245-246Crossref PubMed Scopus (4799) Google Scholar). Because Axin is expressed in most if not all cell types, a murine macrophage cDNA library cloned in the pGADNOT prey vector was screened to identify proteins that can interact with Axin. After co-transformation of pGBT9-Axin-(632–956) with the expression library, 12 colonies out of approximately 50,000 potential transformants survived on synthetic medium lacking histidine (Table I, 1o screen). The growth of these colonies implied that they contained clones encoding proteins that bound to Axin-(632–956) and also suggested the expression of the his reporter gene under the control of a Gal4-responsive promoter in these transformants. The expression of a second Gal4-responsive reporter gene, β-galactosidase, was found in 9/12 of these colonies (Table I, 2o screen). All nine of these clones tested negative for β-galactosidase in the absence of the bait vector pGBT9-Axin-(632–956) (Table I, 3o screen).Table ISummary of identification of Axin-binding proteins by yeast two-hybrid screeningScreening/clone no.1234567891011121°-his+++++++++++++2°-β-Gal+++++++++−−+−3°-No bait plasmid, β-Gal+−−−−−−−−NANA−NA4°-Reconstitution, +Gal4DB−−−−−−−−NANA−NA+Gal4DB-Axin−−+−−−+−NANA+NAThe primary selection was the growth of yeast transformants on histidine−medium (1°), followed by the expression of a second reporter gene (2°), β-galactosidase (β-Gal). The 3° screen was done by losing the bait pGBT9-Axin-(632–956) plasmid in those transformants. Finally, pGADNOT prey plasmids were recovered and cotransformed with the bait vector containing either Gal4DB or Gal4DB-Axin to reconstitute the expression of the β-galactosidase reporter gene (4°). +, positive; −, negative; NA, not applicable. Open table in a new tab The primary selection was the growth of yeast transformants on histidine−medium (1°), followed by the expression of a second reporter gene (2°), β-galactosidase (β-Gal). The 3° screen was done by losing the bait pGBT9-Axin-(632–956) plasmid in those transformants. Finally, pGADNOT prey plasmids were recovered and cotransformed with the bait vector containing either Gal4DB or Gal4DB-Axin to reconstitute the expression of the β-galactosidase reporter gene (4°). +, positive; −, negative; NA, not applicable. The pGADNOT prey plasmids were recovered from these nine transformants. Only three of them exhibited the ability to bind to Axin upon co-transformation with pGBT9-Axin-(632–956) in the yeast two-hybrid system (Table I, 4o screen). Two of the three plasmids contained a 1.9-kb DNA insert and the other a 1.3-kb insert. DNA sequence analysis of the two 1.9-kb clones showed that they were identical and encoded a mouse protein displaying 99% amino acid identity to the α isoform of the catalytic subunit of rat and human serine/threonine protein phosphatase 2A (PP2Ac). 2The nucleotide sequence for the mouse PP2Ac gene has been deposited under accession numberAF076192. The 1.3-kb clone encoded a C-terminal segment of Axin protein (amino acids 831–956 of form 1). These data suggest that Axin binds to PP2Ac and also associates with itself through the C-terminal region. PP2A is a heterotrimeric enzyme consisting of a catalytic subunit (C) associated with a 65-kDa regulatory subunit (A) and a third variable subunit (B) (36Cohen P. Annu. Rev. Biochem. 1989; 58: 453-508Crossref PubMed Scopus (2135) Google Scholar, 37Mumby M.C. Walter G. Physiol. Rev. 1993; 73: 673-699Crossref PubMed Scopus (623) Google Scholar, 38Shenolikar S. Annu. Rev. Cell. Biol. 1994; 10: 55-86Crossref PubMed Scopus (401) Google Scholar). In mammals, the closely related α and β isoforms of PP2Ac are encoded by separate genes but are indistinguishable in function. Axin contains several predicted sites for Ser/Thr phosphorylation (2Zeng L. Fagotto F. Zhang T. Hsu W. Vasicek T.J. Perry III, W.L. Lee J.J. Tilghman S.M. Gumbiner B.M. Costantini F. Cell. 1997; 90: 181-192Abstract Full Text Full Text PDF PubMed Scopus (779) Google Scholar), and Ser/Thr phosphorylation of β-catenin is thought to play a critical role in Wnt signaling (14Miller J.R. Moon R.T. Genes Dev. 1996; 10: 2527-2539Crossref PubMed Scopus (606) Google Scholar,15Yost C. Torres M. Miller J.R. Huang E. Kimelman D. Moon R.T. Genes Dev. 1996; 10: 1443-1454Crossref PubMed Scopus (1009) Google Scholar). This suggested that the Axin·PP2Ac interaction might be biologically significant. Therefore, the ability of PP2Ac to interact physically with Axin was independently examined by an in vitro binding assay (Fig.1). Three different recombinant GST-Axin fusion proteins and control GST protein were bacterially expressed and purified (Fig. 1 A) and were incubated with in vitro synthesized, 35S-labeled PP2Ac. Analysis of protein complexes precipitated with glutathione-Sepharose indicated that PP2Ac bound to a C-terminal region of Axin, amino acids 632–956 (Fig. 1 B, lane 5). However, two fusion proteins lacking the last 146 amino acids, GST-Axin-(421–810) and GST-Axin-(632–810), failed to bind PP2Ac (Fig. 1 B, lanes 3 and 4). To examine the association of Axin and PP2Ac in vivo, T7 epitope-tagged PP2Ac and three different FLAG-tagged Axin proteins were transiently expressed in 293T cells (Fig.2, A and B). PP2Ac was co-immunoprecipitated with FLAG-Axin-(632–956), which contains a C-terminal polypeptide identical to the one used as bait in the yeast two-hybrid screen, but not with FLAG-Axin-(194–475), which includes the RGS domain (Fig. 2 C). The FLAG-Axin-(632–992), derived from form 2 of Axin, also co-precipitated with PP2Ac, suggesting that both isoforms of Axin interact with PP2Ac regardless of the 36 amino acids insertion (Fig.2 C). Similar results were also obtained when cell lysates were first immunoprecipitated with anti-FLAG antibody followed by immunoblotting with anti-T7 antibody (Fig. 2 D). These data agree with the conclusions from yeast two-hybrid and in vitro biochemical analyses, confirming the ability of Axin and PP2Ac to interact in vivo. The interaction of Axin and PP2Ac in various experimental assays raised the question whether Axin binds directly to the PP2Ac subunit or whether it might associate indirectly with PP2Ac by binding to the regulatory A subunit, which itself binds tightly to the catalytic subunit. A co-immunoprecipitated assay was first performed to test whether the regulatory A subunit was present in the Axin·PP2Ac complex. The T7-tagged PP2Ac and HA-tagged PR65 (regulatory A subunit of PP2A) were transiently expressed together with four different Myc- or Flag-tagged Axin proteins in 293T cells (Fig.3, A and B). PR65 only co-precipitated with Axin proteins that contain the PP2A-binding domain, indicating that PR65 can indeed associate with Axin in vivo (Fig. 3 C). Next, the yeast two-hybrid assay was used to analyze further the ability of Axin to bind to PR65. Unlike the catalytic subunit, PR65 failed to show any interaction with Axin in this assay (Table II). We therefore conclude that Axin can bind directly to the PP2Ac catalytic subunit and only indirectly to PR65.Table IIThe regulatory A subunit of PP2A, PR65, does not interact with Axin in the yeast two-hybrid system.BaitPreyβ-Gal filter assayAxin-(632–956)PP2Ac+++PP2AcPR65+++Axin-(194–956)PR65−Axin-(632–956)PR65−Axin-(632–992)PR65−In a control experiment, PR65 was able to interact with PP2Ac, its normal partner, in this assay. +++, dark blue developed after 5 h of β-galactosidase (β-Gal) assay; −, only white color after 12 h of β-galactosidase assay. Open table in a new tab In a control experiment, PR65 was able to interact with PP2Ac, its normal partner, in this assay. +++, dark blue developed after 5 h of β-galactosidase (β-Gal) assay; −, only white color after 12 h of β-galactosidase assay. The cloning of a fragment of Axin by the yeast two-hybrid screen with pGBT9-Axin-(632–956) (Table I) raised the possibility that Axin may form dimers or high order complexes with itself. The self-association of Axin was tested using the co-immunoprecipitated assay (Fig.4). A Myc epitope-tagged Axin-(811–956), which could be distinguished from FLAG-tagged Axin proteins, was transiently expressed in 293T cells (Fig. 4 A). The FLAG-Axin proteins were expressed simultaneously (Fig. 4 B) and tested for co-immunoprecipitation (Fig. 4 C)